TW200808338A - Vasodilator and functional food - Google Patents

Abstract

It is intended to provide a vasodilator having a vascular endothelium-dependent vasodilating action, a preventive agent for arteriosclerosis, and a functional food having a vascular endothelium-dependent vasodilating action which can be continuously used on a daily basis and is excellent in safety. The vasodilator and the functional food of the invention are characterized by comprising, as an active ingredient, at least one of tripeptides of Val Pro Pro and Ile Pro Pro or a protein degradation product containing Val Pro Pro and/or Ile Pro Pro.

Description

200808338 (1) Description of the Invention [Technical Field] The present invention relates to a vasodilator having an endothelium-dependent vasodilating action, and a functional food having the same effect. - [Prior Art] Patients with arteriosclerosis eventually develop myocardial infarction and cerebral infarction. • Ischemic symptoms are the most common cause of death in Japan. Risk factors for arteriosclerosis include hyperlipidemia, hypertension, diabetes, smoking, obesity, hyperuricemia, aging, and stress. These factors J are interlaced to cause vascular disorders. Therefore, even if the risk factors are mild, the risk factors are additive and multiplicative due to the repetition of these risk factors, which increases the risk of ischemic symptoms. However, it is currently known that reducing only one of the above risk factors is not sufficient to prevent the occurrence of arteriosclerosis. For example, in Science of Food 257 ( (1999) p20-25), and Atherosclerosis 1 5 1 ((2000) p5 0 1 - 5 08), it is shown that the concentration of cholesterol in the blood is not related to arteriosclerosis, in Circulation 1 04 ((200 1 ) p239 1 - 2394) ^ Even if the state of hypertension is suppressed, no change in the degree of arteriosclerosis is observed, and in the International Journal of Cardiology 81 ((200 1 ) P107-115), even An angiotensin-converting enzyme inhibitor with enalapril (ENALAPRIL) as an active ingredient was also administered, and no inhibition of arteriosclerosis was observed. Furthermore, from these results, infarctotic ischemic symptoms, such as acute arrhythmia, according to the separate book -4- (2) 200808338 medical progress of circulatory diseases (-state of arts v P3 32-3 34 ) In order to stabilize the blood flow, it is necessary to administer a vasodilator such as sodium sulphate or nitroglycerin, and a diuretic is strong. Therefore, three wins are described in Japanese Patent Publication No. Hei 6-97786 and Japanese Patent Laid-Open Publication No. Hei. Peptide Val Pro Pro and lie have lower blood pressure, can inhibit vasopressin I conversion, and have anti-stress effects. However, according to these results, it is impossible to say that it has anti-atherosclerosis effect, and it cannot be called vasodilator artery. The wall of the hardened arterial tube becomes hypertrophy and gradually loses the ambulatory disease. In recent years, one of the main causes of this symptom is Λ injury and low function. Therefore, it is expected that the vascular endothelium-dependent vasodilatation 51 pulse hardening inhibition effect. SUMMARY OF THE INVENTION The subject of the present invention is to provide a vasodilator having vascular endothelium-dependent blood sales. Another object of the present invention is to provide a function of vascular endothelium-dependent vasodilatation which is excellent in daily valence, and according to the present invention, a vasodilator having at least one peptide of Pro Pro and lie Pro Pro To be effective and in accordance with the present invention, a proteolytic component of a vasodilator Val Pro Pro and/or 116?|*??1* can be provided. Er.2(200 1 ) ί nitrate, ferricyanide, etc. 11-100328 Pro Pro » Activity of the prime 3 The three peptides: role. 〖Sexual type 1 tube endothelium agent has a dynamic expansion effect and safe food. Contains Val points. Further, the conjugate is effective. -5-200808338 (3) Further, according to the present invention, a vasodilating functional food containing at least one peptide of Val Pro Pro and lie Pro Pro as an active ingredient can be provided. Further, according to the present invention, it is possible to provide a vasodilating machine-energy food containing a protein-decomposing product of Val Pro Pro and/or lie Pro Pro as an active ingredient. According to the present invention, at least one peptide of Val Pro Pro and lie Pro Pro for producing a vasodilator or a vasodilating functional food, and a protein decomposition product containing Val Pro Pro and/or lie Pro Pro are provided. Use. The vasodilator and the functional food of the present invention contain Val Pro Pro and/or lie Pro Pro derived from casein of animal milk, and further include a protein decomposition product containing at least one of these peptides as an active ingredient. It has good safety and has an endothelium-dependent vasodilating effect, and it can be taken daily when it is used as a functional food. φ Therefore, the vasodilator of the present invention can effectively ensure the smooth flow of blood in the ischemic symptoms such as myocardial infarction and cerebral infarction, and not only can cause the contraction due to age and lifestyle diseases. The 'vascular vascular relaxation, can also be expected to prevent atherosclerosis prevention, blood flow disorders related to the shoulders, cold hands and feet, thrombosis. The functional food of the present invention can be used as a health food and a specific health food for the treatment of various diseases and symptoms such as fifty shoulders, cold hands and feet, and blood clots associated with vasodilation and blood flow disorders. The invention is explained in more detail below. -6- (4) 200808338 The vasodilator and functional food of the present invention contains Val Pro Pro and/or lie Pro Pro (hereinafter referred to as VPP and IPP, respectively), or contains these peptides. At least one of the protein degradation products is an active ingredient. "It is also possible to add pharmacologically acceptable salts to the aforementioned three peptides, such as inorganic salts such as hydrochlorides, sodium salts and phosphates, and citrates, maleates, fumarates, a salt such as an acid salt such as a tartrate or a lactate. The method for preparing the tripeptide is, for example, a peptide or protein having a VPP and/or IPP amino acid sequence, which is decomposed by microbial fermentation. In addition to the purification method or the enzyme hydrolysis method, it can also be obtained by a synthesis method. For details, refer to the aforementioned Patent Documents 1 and 2. The active ingredient used in the present invention is a VPP and/or IPP amine group. a peptide or protein of an acid sequence, which is fermented by a microorganism, and then a fermentation product containing at least one peptide of VPP and IP P or a purified product thereof, or a VPP and/or IPP amino acid sequence a peptide or protein which is decomposed by an enzyme Φ and which contains a hydrolyzate of at least one peptide of VPP and IPP or a purified product thereof. The effective dosage of the vasodilator of the present invention is a single administration, and each effect is obtained. The amount of human daily three peptides is generally 1 0 // g 1 〇g is better, about 1 mg ~ 1 g is better. The administration of vasodilators can be adjusted according to the symptoms of the disease. It is suitable for single and continuous non-oral administration of acute symptoms. For chronic and preventive It is preferred to use oral exhalation for more than 30 days. The vasodilator of the present invention can be administered orally and non-200808338 (5) by oral administration. The topical, transdermal, medullary, subcutaneous, intradermal, intraperitoneal, intrathoracic, and intraspinal canal can also be administered directly to the affected part. The vasodilator of the present invention can be administered according to the above formula ^ The formulation may be in the form of a preparation, for example, a tablet, a nine-dose, a dose, a soft capsule, a microcapsule, a powder, a granule, a liquid, an emulsion, etc. φ When the formulation is as described above, Suitablely used as a carrier, adjuvant, excipient, patch, preservative, agent, binder, pH adjuster, buffer, viscosity enhancer, gel preservative, antioxidant Etc., generally accepted in the form of unit dosage in the formulation implementation The functional food of the present invention is a health food health food or the like which can label or promote various diseases related to blood flow and blood flow disorders, etc. • In order to obtain the effect, in particular, vasodilation and accompanying thereof The amount of intake of the disease, considering the functional food, can be used continuously and can be taken for a long time, the amount of the three peptides per day is generally 1 ο μ " preferably, about 1 mg ~ 1 g. For the daily intake times, the amount of each of the functional foods can be reduced with the amount of the above-mentioned three peptides. The intake time of the functional food of the present invention is not particularly limited to the improvement of chronic symptoms and In the case of prevention, long-term ingestion is used to obtain the above-mentioned effects. Generally, it is taken for more than 30 days, especially for 3' intrapulmonary, and the method is to allow the hardening of the hard capsule suspension to be used as a stabilizer, and the required tube expansion and Specific disease improvement 曰 regularity g ～1 0g The above calculation, and, the use is good. It is better for daily intake of 12 -8- 200808338 (6) months. The functional food of the present invention can be produced by mixing a peptide which is an active ingredient or a food material containing the three peptides, and the like, and can be produced as a food or drink. It is made into candy, yoghurt, milk thistle, dairy, wine - water, powder or granulated food, capsule food, various camping foods and other forms. It is also possible to use in the functional food of the present invention φ various additives which can be generally added to foods. [Embodiment] EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is intended to be limited to these examples. Synthesis Example VPP and IPP were synthesized by the following organic chemical synthesis method. The peptide synthesis device (PSSM-8 type) manufactured by the company was synthesized in g rows. The solid phase support is 2-Chlorotrityl §! Vinyl resin, which is a methoxycarbonyl group (hereinafter referred to as Fmo amine-based proline-bonded resin 5 Omg (manufactured by Shimadzu Corporation) g SynProPep Resin). According to the amino acid sequence, Fm〇c-lie and Fmoc-Fmoc-Val of Fmoc-based protective limbs of each | moi are respectively described in accordance with the sequence of peptides according to the sequence of peptides. Morphology. Cooling strengthening or mixing is not limited to the use of the island phase method of polyphenylene) protection book trademark 100 β Pro and its anti--9 - 200808338 (7) should win the peptide bonding resin. Next, the peptide-bonding resin was suspended in 1 ml of the reaction liquid A (10% acetic acid, 10% capacity trifluoroethanol, 80% capacity dichloromethane), and allowed to react at room temperature. After the peptide was cleaved from the resin ^ in minutes to 60 minutes, the reaction solution A was filtered through a glass filter. After removing the filtrate and the solvent under reduced pressure, 1 ml of the reaction solution (82.5% capacity of trifluoroacetic acid, 3% capacity of sulphide, 5% of pure water, 5% of the volume of fenium φ) was added. Aromatic thioether, 2.5% by volume of ethanedithiol, 2% by volume of thiophenol) was reacted at room temperature for 6 hours to remove the branched protecting group. Further, 10 ml of water-free methanol was added to precipitate the peptide, which was separated by centrifugation at 3000 rpm for 5 minutes. The precipitate was washed several times with anhydrous methanol, then passed with nitrogen and dried. The whole amount of the unpurified synthetic peptide obtained in this manner was dissolved in 2 ml of a 0.1 N aqueous hydrochloric acid solution, and purified by HP LC using a C18 analytical column under the following conditions. High-pressure pump: L6200 Intelligent .Pump (Hitachi), # Detector: L4000UV detector (Hitachi), absorbing column at 215nm UV: gBondasphere 5μ C18 (made by Watershe), mobile phase: Liquid A was 0.1% by weight of TFA aqueous solution, and Liquid B was acetonitrile (B/A + B ) *100 (%) containing 0.1% by weight of TFA: 〇-> 40% (60 minutes), flow rate: 1 ml/min. The dissolution fraction showing the maximum absorption was separated, and the synthetic peptides VPP and IPP of the target obtained by freeze-drying were 5.7 mg and 6.5 mg, respectively. The purified peptide was analyzed by a fully automated protein primary structure analyzer (PPSQ-10, manufactured by Shimadzu Corporation), starting from the N-terminal of the peptide, and then using an amino acid analyzer (800 series, -10- (8) (8) 200808338 曰本分光公司) After analyzing the results, the confirmation is the same as the design. Example 1 (Vasodilation test) The thoracic aorta of Wistar rats was removed and excised 2 mm wide to form an aortic annulus. A Magnus device (manufactured by Labo-support, product name Micro Tissue Organ Bath MTOB·1Z) was placed on the ring to give a fixed tension to balance. The contraction response of the aortic annulus was confirmed by 50 mM KC1. Next, the aortic annulus was contracted with 1 mM of phenylephrine, and the vasoendothelial-dependent expansion reaction was observed by the 1#M choline choline in the sample which was stabilized and contracted, and it was confirmed that it maintained the vascular endothelial function. Then, after the aorta was contracted by 1 / z phenylephrine, the VPP and sputum prepared in the synthesis example were added and the concentration was increased by 10 times from 1〇·9Μ, and the tension change from the aortic ring was observed. Expansion reaction. In addition, the amount dependence of the expansion reaction was also reviewed. As a result, the vasodilatation reaction caused by VPP and sputum was observed from a concentration of 1 mM, and an excessive vasodilatation reaction was clearly observed at 1 OmM. The result is shown in Fig. 1. (Endothelium-dependent confirmation test for VPP and vasodilatation of sputum) To confirm the association between vasodilatation of VPP and IP P and vascular endothelium, the vascular endothelium is physically removed by conventional methods. For comparison, the VPF was subjected to the same vasodilation test as above, and the vasodilation reaction was attenuated by the removal of the endothelium. Therefore, it was found that the vasodilation of VPP and IPP was extremely dependent on the vascular endothelium. The result is shown in Fig. 2. Comparative Example 1 The same vasodilation test as in Example 1 was carried out by substituting the VPP and IPP of the tripeptide with the same concentration of amino acid valine (Val) or valine (Pro). As a result, the 10 mM expansion reaction was not observed in Val and Pro. The result is shown in Fig. 1. From the above results, it is known that the vasodilating action of VPP and IPP is active only in the form of a peptide. In the case of the vasodilating medicinal composition and the vasodilating health food composition, various peptides derived from milk proteins are hydrolyzed as peptides as an active ingredient. Since the alkyl acid sequence containing the VPP and IPP sequences is known to be present in cold-casein and /C-casein, the peptide obtained according to the production example described in the publication is confirmed to include VPP and IPP. In the production example, a commercially available skim milk powder containing a protein material of /3 -casein and /c -casein was tested. That is, 1 kg of skim milk powder was suspended in 2 liters of warm water, and after adjusting the pH to 7.5, 40 g of heat-resistant enzyme (manufactured by Daiwa Kasei Co., Ltd.) was added, and the reaction was carried out at 50 ° C for 16 (10) 200808338 hours. After the reaction is completed, the enzyme is inactivated by heating at 1 〇 〇 °C for 1 〇 to produce a hydrolyzed peptide. After analysis by high performance liquid chromatography mass spectrometry (LC/MS), VPP and hydrazine were not detected therein. According to the above results, it is clear that the vasodilation reported in Japanese Patent Publication No. 2004-2443 59 is caused by components other than VPP and sputum.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the results of a blood vessel expansion test performed in Example 1 and Comparative Example 1. ® 2 is a graph showing the results of an endothelium-dependent confirmation test of the vasodilation reaction performed in Example 1.

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Claims (1)

200808338 (1) X. Patent application scope L A vasodilator containing at least one peptide of Val Pro Pro and lie Pro Pro as an active ingredient. 2. A vasodilator comprising a protein decomposition product of Val Pro Pro and/or lie Pro Pro as an active ingredient. 3. A functional food having vasodilating action, which comprises at least one peptide of Val Pro Pro and lie Pro Pro as an active ingredient. 4. A functional food having a vasodilating action, which comprises a Pro Protease of Val Pro Pro and/or 116?1:???〇 as an active ingredient.